Qualitative and Quantitative Analysis of the Major Bioactive Components of Juniperus chinensis L. Using LC-QTOF-MS and LC-MSMS and Investigation of Antibacterial Activity against Pathogenic Bacteria

Plants in the genus Juniperus have been reported to produce a variety of chemical components, such as coumarins, flavonoids, lignans, sterols, and terpenoids. Here, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were applied to qualitatively and quantitatively analyze the major bioactive components in an ethanolic crude extract from the leaves of Juniperus chinensis L., which grows naturally in Korea. In addition, the antibacterial activity of the crude extract against pathogenic bacteria was investigated. Using LC-QTOF-MS analysis, we identified ten compounds, of which six were confirmed to be flavonoid and lignan-based components as the major bioactive components, i.e., isoquercetin, quercetin-3-O-α-l-rhamnoside, hinokiflavone, amentoflavone, podocarpusflavone A, and matairesinoside. Among them, a quantitative analysis performed using LC-MS/MS revealed that the levels of quercetin-3-O-α-l-rhamnoside and amentoflavone in the crude extract were 203.78 and 69.84 mg/g, respectively. Furthermore, the crude extract exhibited potential antibacterial activity against 10 pathogenic bacteria, with the highest antibacterial activity detected against Bordetella pertussis. Thus, further studies of the leaf extract of J. chinensis L. must be carried out to correlate the compounds present in the extract with the antibacterial activity and elucidate the mechanisms of action of this extract against bacteria.


Introduction
Juniperus chinensis L. is known as Chinese juniper and is commonly found in many regions in Asia, including China, Taiwan, Myanmar, Japan, Malaysia, and Korea [1]. The genus Juniperus is considered as an important source of medicinal plants that are rarely used in traditional medicine. Various Juniperus species have been reported to produce a variety of chemical components, such as coumarins, flavonoids, lignans, sterols, and terpenoids [2], with a wide range of medicinal properties, such as antimicrobial, antioxidant, antitumor, and anticancer effects [3][4][5][6].
Flavonoids or bioflavonoids are a class of polyphenolic secondary metabolites that are found in plants and fulfill many functions [7]. The basic structure of these compounds consists of a general structure of 15 carbons with two phenyl rings and a heterocyclic ring, termed diphenylpropane (C6-C3-C6) skeleton [8]. Flavonoids are especially well known as having antibacterial properties against many pathogenic microorganisms. Recently, several investigations were performed regarding the antimicrobial activities, also summarizing the probable relationships between the chemical structures and antimicrobial activities [8]. These compounds exert their antibacterial effects via a mechanism mainly targeting the cell membrane, which likely involves the inhibition of attachment and biofilm formation, the inhibition of phospholipid bilayer formation, the inhibition of the respiratory chain, ATP-synthesis-mediated damage to the energy metabolism, etc. [7,9,10]. Lignans are a class of polyphenolic secondary metabolites that are found in plants and are precursors to phytoestrogens. These compounds have a general basic structure of nine carbons consisting of two phenylpropane (C6-C3) skeletons [11] and have been reported to have anticancer, antioxidant, antimicrobial, anti-inflammatory, and immunosuppressive activities [12][13][14].
In the present study, we qualitatively analyzed the bioactive components of a crude extract of J. chinensis L. using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Among them, quantitative analysis was performed for those with a mass spectrum, fragmentation pattern, and retention time which matched the reference compounds based on liquid chromatographytandem mass spectrometry (LC-MS/MS). Furthermore, the antibacterial activity of the crude extract against 10 pathogenic bacteria was evaluated.

Chemical Profiling of Juniperus chinensis L.
An ethanolic crude extract of the leaves of J. chinensis L. was analyzed and characterized using UPLC-QTOF-MS in electrospray ionization (ESI) negative ion mode, because more information and a higher fragmentation could be obtained in this ion mode compared with positive ion mode. We investigated various physiologically active component peaks in the base peak ion chromatogram, as shown in Figure 1. Ten components were tentatively identified after a library search using Waters' UNIFI software (version 1.9, Milford, MA, USA) and the ChemSpider online database of isolated components under chromatographic conditions. All components exhibited a mass error below 5 mg/kg (Table 1, Supplementary Figure S1). Among them, the components corresponding to peaks 4 and 7 were identified as quercetin-3-O-α-L-rhamnoside and amentoflavone, respectively, based on reference compounds with mass spectra with identical patterns (Figure 2 [33,34]. Of the 10 compounds identified in this study, six were confirmed to be flavonoid and lignan-based components which have diverse physiological activities according to the literature.

Quantitative Analysis of the Reference Compounds
The two reference compounds, quercetin-3-O-α-L-rhamnoside and amentoflavone, were analyzed using UPLC-Xevo TQ-S micro MS/MS in ESI mode. The analytical conditions of the instrument were set so that the retention time of the analyte was the same on the chromatograms of the standard solutions and the sample solutions. The ion ratio was calculated as the peak area ratio between a less intense ion and a more intense ion. The reference ion ratio value was calculated as the average of the ion ratios of the calibration solutions. All samples met the instrument analytical conditions described above (not shown). Moreover, the quercetin-3-O-α-L-rhamnoside standard calibration revealed a coefficient of determination (R 2 ) of 0.9991 with good linearity, ranging from 0.05 to 1 ng. The amentoflavone standard calibration revealed a coefficient of determination (R 2 ) of 0.9955 with good linearity, ranging from 0.25 to 2.5 ng. Overall, these results indicated that the methods were sufficiently validated to identify the two reference compounds in the samples, as a result of the standard curves of quercetin-3-O-α-Lrhamnoside and amentoflavone and the measurement of the total compound content ( Table 2). The total contents (weight) in the ethanolic extract of quercetin-3-O-α-Lrhamnoside and amentoflavone were 203.78 and 69.84 mg/g, respectively.  Peak 3 (observed RT: 4.84 min; formula: C 21 H 20 O 12 ) was identified as isoquercetin, as reported in [15,16]. The precursor ion of this component was detected at m/z 463.0878 [M − H] − ion, with a fragment ion at m/z 300.0267 [463-C 6 H 12 O 5 ] − with the main peak. Isoquercetin has been reported to inhibit the oxidative stress effect of multiple carcinogens [21,22]. Peak 4 (observed RT: 5.34 min; formula: C 21 H 20 O 11 ) was identified as quercetin-3-O-α-L-rhamnoside, as reported in [16][17][18] [33,34]. Of the 10 compounds identified in this study, six were confirmed to be flavonoid and lignan-based components which have diverse physiological activities according to the literature.

Quantitative Analysis of the Reference Compounds
The two reference compounds, quercetin-3-O-α-L-rhamnoside and amentoflavone, were analyzed using UPLC-Xevo TQ-S micro MS/MS in ESI mode. The analytical conditions of the instrument were set so that the retention time of the analyte was the same on the chromatograms of the standard solutions and the sample solutions. The ion ratio was calculated as the peak area ratio between a less intense ion and a more intense ion. The reference ion ratio value was calculated as the average of the ion ratios of the calibration solutions. All samples met the instrument analytical conditions described above (not shown). Moreover, the quercetin-3-O-α-L-rhamnoside standard calibration revealed a coefficient of determination (R 2 ) of 0.9991 with good linearity, ranging from 0.05 to 1 ng. The amentoflavone standard calibration revealed a coefficient of determination (R 2 ) of 0.9955 with good linearity, ranging from 0.25 to 2.5 ng. Overall, these results indicated that the methods were sufficiently validated to identify the two reference compounds in the samples, as a result of the standard curves of quercetin-3-O-α-L-rhamnoside and amentoflavone and the measurement of the total compound content ( Table 2). The total contents (weight) in the ethanolic extract of quercetin-3-O-α-L-rhamnoside and amentoflavone were 203.78 and 69.84 mg/g, respectively.

Antibacterial Analysis
The antibacterial activities of the ethanolic crude extract of the leaves of J. chinensis L. were evaluated against pathogenic strains. The ethanolic crude extract of J. chinensis L. exhibited antibacterial activity against all strains tested here ( Table 3). The inhibition zones were similar to or lower than those of antibiotics. Our results showed activity for both grampositive and gram-negative pathogens similar to those observed in the literature [2,35,36]. For E. coli and S. aureus, antibacterial activity was observed among various Juniperus leaves and fruits [35,36]. Ennajar M et al. showed intense activity for K. pneumoniae, while our results showed only low activity [37]. On the other hand, the crude extract of J. chinensis L. exhibited strong antimicrobial activity against Bordetella pertussis NCCP13671.

Plant Material
The leaves of Juniperus chinensis L. were collected on 6 November 2018 from Imhyeonri, Eosangcheon-myeon, Danyang-gun, Chungcheongbuk-do, South Korea. The National Institute of Biological Resources (NIBR) performed the botanical identification of the plant materials. Specimens and materials of J. chinensis L. were deposited in the Wildlife Natural Products Bank at the NIBR, with NIBR numbers NIBRVP0000725557 and NI-BRGR0000611643, respectively.

Preparation of the Plant Extract
The leaves of J. chinensis L. were collected to prepare the ethanolic extract. The leaves were dried in an oven (HB-503SF, HANBAEK) at 40 • C for 48 h and then ground to a fine powder to pass through a 20-mesh sieve. The powder was extracted with 70% ethanol (Sigma-Aldrich, Saint Louis, MO, USA) at four times the volume (1:4, v/v). The mixture was left at room temperature for 72 h, centrifuged at 4000× g for 10 min (VARISPIN 15R, CRYSTE), and filtered using filter paper (Whatman No. 2, 4.25 cm diameter). Moreover, the solvent was concentrated by rotary evaporation (Rotavapor R-100, Buchi, Flawil, Switzerland). The crude extract was stored at −80 • C in a freezer until it was used for analysis of the major bioactive components and antibacterial activity.

Qualitative Analysis Using Ultra-High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry
The chemical profiling analysis of the crude extract from J. chinensis L. was performed using Ultra-High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UPLC-QTOF-MS) in negative modes using an ACQUITY TM UPLC-Xevo GS-XS Q-TOF (Waters) instrument equipped with an ACQUITY UPLC ® BEH C18 column (100 × 2.1 mm, 1.7 µm; Waters, Milford, MA, USA) at 40 • C. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.4 mL/min, and the injection volume was set at 1 µL. At this time, the injected sample was diluted to 2500 mg/L level with LC grade methanol. The mobile phase was a gradient: 0-1 min, 5% solvent B; 1-20 min, 100% solvent B; 20-20.30 min, 100% solvent B; 22.30-22.40 min, 5% solvent B; and 22.40-25 min, 5% solvent B. Full-scan mass spectra were acquired to detect the mass-to-charge ratio (m/z) in the range of 50-1500. The detector conditions were optimized as follows: the source temperature was set at 120 • C with a capillary voltage of 2.5 kV. Data acquisition and analysis were controlled using the traditional medicine library in the Waters' UNIFI software (version 1.9, Milford, MA, USA). First, the identification of the components was performed by checking the allowable chemical formula calculated for the mass error range ± 5 ppm and setting it to fragment tolerance 10 mDa to check the mass pattern. In addition, if the fragment ion pattern did not match, it was finally identified using the Chemspider (http://www.chemspider.com/, accessed on 27 September 2022) online database.

Reference Compounds and Preparation of the Standard Solution
Reference compound quercetin-3-O-α-L-rhamnoside (98% purity) was purchased from LGC standard, whereas amentoflavone (98% purity) was purchased from Sigma-Aldrich. The reference compounds were prepared by dissolving them in methanol at a final concentration of 1000 mg/L. The standard solutions were diluted to obtain calibration curves with five points in the concentration range of 0.025-0.5 and 0.05-0.5 mg/L, respectively.

Antibacterial Analysis
The antibacterial activity of the crude ethanol extract from J. chinensis L. was evaluated against various pathogenic bacterial strains using a modified disk diffusion method [38]. This study used 10 pathogenic bacteria strains deposited in NCCP or ATCC to conduct the antibacterial analysis: Escherichia coli KCTC 2617, Salmonella enterica serovar Enteri-tidis NCCP 14546, Streptococcus mutans KCTC 3065, Staphylococcus aureus NCCP 14560, Acinetobacter baylyi ATCC 33305, Klebsiella pneumoniae NCCP 16052, Bordetella pertussis NCCP13671, Moraxella catarrhalis ATCC 43628, Staphylococcus pyrogenes NCCP14783, and Streptococcus pneumoniae NCCP 14774. Each bacterial pathogenic strain was grown on suitable media at 30 • C-37 • C for 20 h. E. coli, S. aureus, and K. pneumoniae were grown on nutrient agar; S. enteritidis on tryptic soy agar; S. mutans, A. baylyi, M. catarrhalis, and S. pyrogenes on brain heart infusion agar; B. pertussis on Bordet-Gengou agar; and S. pneumoniae on sheep blood agar. To carry out the disc diffusion test as an antimicrobial disc susceptibility test according to the guidelines of the Clinical & Laboratory Standard Institute, each agar plate medium was inoculated with pathogenic bacteria at a concentration of 1-2 × 10 8 CFU/mL. Sterilized paper discs (8 mm) were placed on the agar, and 10 µL of the crude plant extract was used to impregnate the discs. The extracts were dissolved in DMSO and ethanol (1:1, v/v) at a concentration of 20 mg/mL. The culture was incubated at 30 • C-37 • C for 24 h, and the diameters of the inhibition zones that formed around each disk were measured in millimeters. The positive control consisted of the appropriate antibiotic for each pathogen, i.e., ampicillin (2.5 mg/mL) for E. coli, S. enteritidis, S. mutans, K. pneumoniae, B. pertussis, and S. pneumoniae; gentamycin (2.5 mg/mL) for A. baylyi; penicillin (2.5 mg/mL) for M. catarrhalis and S. pyrogenes; and kanamycin (50 mg/mL) for S. aureus. In contrast, the negative control consisted of DMSO and ethanol (1:1, v/v). Each experiment was performed in triplicate.

Conclusions
In the present study, various chemical components were identified in an ethanolic crude extract of the leaves of J. chinensis L., which grows naturally in Korea. We identified 10 flavonoid and lignan-based molecules via LC-QTOF-MS analysis as the major bioactive components, including isoquercetin, quercetin-3-O-α-L-rhamnoside, hinokiflavone, amentoflavone, podocarpusflavone A, and matairesinoside. Among them, quercetin-3-O-α-L-rhamnoside and amentoflavone were quantitatively analyzed using LC-MS/MS; they existed in the crude extract at 203.78 and 69.84 mg/g, respectively. Moreover, the ethanolic crude extracts showed overall inhibitory activities against all Gram-positive and Gram-negative pathogenic bacteria tested, with the highest activity observed against Bordetella pertussis. Based on these results, we concluded that the J. chinensis L. extract showed antibacterial activity against various pathogens. However, we did not directly check if the 10 components identified here had antibacterial activity. Therefore, further studies are required to assess the antibacterial activity of the compounds isolated from J. chinensis L.